1. Field of the Invention
This invention relates to an improvement in enantiomeric separation and detection by means of a chiral reagent and high performance liquid chromatography (HPLC). More specifically, it relates to an optically active fluorenyl chloroformate which forms stable diastereomeric carbamates with primary and secondary amines in a quantitative way. This product can be detected by fluorimetry or by absorptiometry after separation on an HPLC column.
2. Description of the Background Art
The separation of optically active amino-containing substances is of great importance in biological research and pharmaceutical chemistry. These substances are common in pharmaceuticals and many of these substances are enantiomers occurring as racemic mixtures. In many cases the biological activity of these enantiomers is attributed to the conformation of one particular optically active antipode. Therefore, it is very important to distinguish or resolve the enantiomers of such substances.
For quantitive purposes, it is desirable to use chromatographic methods to separate these substances. These methods offer many advantages including the utilization of small sample sizes, simple pretreatment procedures, and high sensitivity of detection.
Since the first chromatographic separation of optically active isomers was performed in 1951 with a paper chromatography technique, a great number of such methods have been introduced. Some of these methods have recently been reviewed in books including: Souter, R. W. "Chromatographic Separation of Stereoisomers"; CRC Press, Boca Raton, 1985; and Jacques, J. et al., "Enantiomers, Racemates and Resolutions"; John Wiley & Sons; New York, 1981.
The resolution of enantiomers requires the intervention of a chiral agent. Chromatographic resolving methods have been developed in two major directions:
1. Direct resolution of enantiomers in columns with either a chiral stationary phase (CSP) or a chiral component in the mobile phase; and
2. Indirect resolution of formed diastereomers after derivatization with a chiral reagent.
The liquid chromatographic direct separation of enantiomers on chiral stationary phases has received a great deal of attention. Currently, there are some commercially available chiral columns. Recent efforts have focused on the feasability of making a chiral stationary phase for separating as many different types of chiral substances as possible. Still very few amine and amino acids are available that have been directly resolved on chiral columns of any type, without previous derivatization.
Tamegai et al., J. Liq. Chromatography, 1979, 2, 1229 have reviewed methods based on indirect resolution of enantiomers by derivatization with a chiral reagent to form diastereomers which are resolved on conventional columns. Furukawa et al., Chem. Phar. Bul 1975, 23, 1623 were the first to separate enantiomeric amino acids using readily available (+)-10-camphor sulphonyl chloride as a chiral reagent. The carbonyl residue was then transformed in a second step into the p-nitrobenzylester which resulted in derivatives sensitive to UV-detection.
The most common chiral reagents for selective amine function derivatization are those based on isothiocyanate which give UV sensitive thiourea derivatives with primary and secondary amines. The reaction is effected under weakly basic conditions for about 20 minutes at room temperature without formation of by-products.
The most successful reagent of this kind is 2,3,4,6-tert-o-acetyl-p-d-gluco-pyranosyl isothiocyanate (GITC), which is used for both amino acids and amines. The resulting thiourea derivatives of most protein amino acids can be resolved with this reagent on a conventional, reversed phase column within two hours. The resolution obtained is preferably due to the lipophilic nature of the sugar residue combined with conformational rigidity.
Fluorescence chiral derivatization regents for amino containing compounds are rare and only reagents based on 0-phthalaldehyde combined with chiral thiols (OPA) are reported, JP 60 38, 652 (85 38, 652). The different chiral thiol compounds which have been used are N-acetyl-L-cysteine and acetyl-L-cysteine. These compounds yield diastereomeric isoindols with primary amino groups. The reaction is selective and completed within a minute at room temperature in a buffered water mixture without racemization. The formed diastereomers are, however, not stable.
These are some essential and desirable conditions such a reagent should have.
1. It has to be optically pure.
2. It has to contain a chromophore or a fluorophor.
3. The reaction conditions should be mild, otherwise the risk of potential racemization will increase.
4. The reagent should react with primary and secondary amino groups.
5. The resulting diastereomers should be stable.
6. The resulting diastereomers should separate on conventional HPLC columns.
7. The method should be applicable on a preparative scale.
8. The method should be feasible to automate.
There has not been a disclosure or suggestion of a chiral reagent combined with a method which effectively derivatize, detect, and separate racemic mixtures of primary and secondary amino-containing compounds.